A  i  9.3;^ 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BDREAD  OF  PLANT  INDUSTRY     BULLETIN  NO.  225 

B.  T.  GAL1  "V.  IY,  (  S    eau. 


A  SPOT  DISEASE  OF  CATLTFL 


LUCIA    Mel  II  I  OCH, 
Scientific  Assistant,  I  t  of  Plant  I'nibology. 


[881  i:i>   AtJGl  ST  29,  1911. 


f     ».      t  * 


DEPOSITORY 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1911. 


BUREAU  OF  PLANT  INDUSTRY. 


< "hh j  'of Bureau,  Beverly  T.  Galloway. 
Assistant  Chief  of  Bureau ,  William  A.  Taylor. 
Editor.  J.  E.  Rockwell. 
Chitf  Clerk,  James  E.  Jones. 


Laboratory  or  Plant  Pathology. 

SCIENTIFIC  STAFF. 

Erwin  F.  Smith.  Pathologist  in  Charge. 
R.  E.  B.  McKenney,  Special  Agent. 
Florence  Hedges,  Assistant  Pathologist. 
A.  W.  Giampietro,  Assistant  Physiologist. 
Nellie  A.  Brown,  Lucia  McCulloch,  and  Mary  ^Catherine  Bryan,  Scientific  Assistants. 

225 
2 


LETTER  ()Y  TRANSMIT)  \L 


['.  S.   I  >EP  \i:i  mi.\  i   in    A.GRK  mi 

Bi  reai  "i   Plant  I  ndustry, 

Ofi  [<  :    OF  in i:  Cini'.i 
Washington,  P.  (  ..    May  81,  nil. 
Sir:  I  have  the  honor  to  transmit  herewith  a  manuscripl  entitled 
"A  Spol  Disease  of  Cauliflower,"  l>\  Mi  is.  Lucia  McCulloch,  scientific 
assistant  in  tin'  Laboratory  of  Plant  Pathol  d  recommend  its 

publication  as  Bulletin  No.  225  of  the  series  of  this  bureau. 

This  paper  deals  with  a  disea  e  which  is  3hown  to  Ik'  of  bacterial 
origin  and  which  has  no)  been  reported  hitherto. 

The  investigations  of  tins  disease  have  been  carried  out  according 
to  the  advice  and  suggestions  of  Dr.  Erwin  V.  Smith. 
Respectfully, 

Ww    A.  T  UXOR, 
Acting  '  Tiiefof  Bureau. 
lion.  .)  \mi>  V\  ii  30 

S( in  tary  oj  . Ygriculiun  . 


Digitized  by  the  Internet  Archive 
in  2013 


http://archive.org/details/spotdiOOunit 


CONTENTS 


Page. 

i ii  lion 7 

hi",  nl                    7 

ption  of  the  cauliflower  leaf-spol  organism 

Morpholog) 

Reaction  to  stains 10 

t  'ultural  characters 10 

10 

r  stabs L0 

Agar  Maul- LO 

Bei  i  b  •  i ill' in II 

A  id  bouillon II 

Ukaline  bouillon II 

Bouillon  \\  iili  mm  limn  chlorid II 

Beei  bouillon  over  chloroform H 

Brum II 

(  ohn'e  solution II 

Fermi's  solution II 

Nutrient  gelatin 12 

l.i  i  in  us  milk 1- 

Milk 12 

l    i  bii    !          olution 12 

Other  i  haracti  ri  tics L2 

Fermentation  tubes 12 

Ammonia  production 12 

Ml  ral'~ 13 

I    dol r 13 

Bydr in  sulphid L3 

Tempera!  ure  relations 13 

Thermal  death  point I" 

Optimum  temperature 13 

Maximum  temperature. .; 13 

Mini  in  ii  i                      1 1  ure 13 

Effect  of  desiccation L3 

Biinlighl L3 

I :  freezing 13 

\  iialiiv  "ii  culture  media I  l 

i|>  number I  l 

hi M 

Latin                  II 

Summary I" 


ILLUSTRATIONS. 


Page. 
Plate  I.  Upper  surfaces  of  cauliflower  leaves,  showing  natural  infection  with 

Bacterium  macul  icol  urn 1  (i 

II.  Under  surfaces  of  cauliflower,  showing  midribs  spotted  by  natural 

infection  with  Bacterium  maculicolum ]P> 

III.  Under  surfaces  of  cauliflower  leaves  from  hothouse,  inoculated  v\  ilh 

Bacterium  maculicolum 16 

225 

6 


B.  P.  1 


V  SPOT  DISEASE  OF  CAULIFLOWER, 


INTRODUCTION. 

In  April  and  again  in  May,  1909,  diseased  cauliflower  plants  were 
senl  to  the  Laboratory  of  Plant  Pathology  from  a  farm  in  southeast- 
ern Virginia.  In  both  lots  the  leaves  were  closely  covered  with 
brownish  to  purplish  gray  spots  I  to  3  millimeters  in  diameter  (PI.  I). 
There  were  also  larger  diseased  area-  due  to  the  coalescing  of  spots. 
All  parts  of  the  leaves  were  affected.  "Where  the  midribs  and  veins 
were  badly  attacked  the  tissues  had  contracted,  giving  a  puckered 
appearance  to  the  leaves  (PI.  II).  From  the  spot-  a.  bacterium  was 
secured  in  pure  cultures  1>\  means  of  petri-dish  poured  plates  on  agar, 
and  subcultures  from  colonies  thus  obtained  were  used  for  inoculating 
healt  by  cauliflower  plants. 

The  cauliflower  heads  from  the  same  plants  were  noi  iii  good  con- 
dition, but  no  success  attended  the  efforts  to  secure  from  them  the 
same  kind  of  bacterium  that  was  found  in  the  leal'  spots. 

Whal  appears  to  he  the  same  disease  was  also  received  once  on 
cauliflower  from  Florida. 

INOCULATIONS. 

All  inoculations  were  made  by  spraying  the  plant-  with  pure  cul- 
tures of  the  bacterium  suspended  in  water  (24  to  4S  hour  agar  -hints 
washed  oil'  in  sterile  water'.  Young,  healthy  cauliflower  plants  6  to 
10  inches  high  were  used,  some  being  kept  in  infection  cages  and 
some  merely  on  a  bench  in  the  greenhouse. 

The  infection  shows  first  on  the  lower  surface  of  the  leaves  as 
sunken  water-soaked  spots.1  These  are  visible  on  the  third  day 
after  inoculation.  In  I  or  5  days  the  spots  are  dark  purplish-gray 
and  -how  on  both  surface-.  |n  transmitted  light  the  centers  are 
thin,  almosl  coli.iT'—.  and  surrounded  1>\  a  dark  border.  In  size 
they  vary  from  mere  point-  to  spots  l  .">  millimeter-  in  diameter.  In 
shape  they  are  irregularly  angled;  the  spread  of  the  disease  appears 
to  be  stopped  or  hindered  by  the  veins  of  the  leaf.     The  individual 

1  Somo  infections  from  old  cultures  thai  bad  probably  lost  much  of  their  virulence  were  first  Indicated 
ciftei  •       •  by  tiny  water-soaked  elevations.    As  the  disease  progressed  the  tissues  collapsed  and 

a. sunk  ilted. 

7 


b  A    SPOT    DISEASE    OF    CAULIFLOWER. 

spots  do  not  become  more  than  2  to  3  millimeters  in  largest  diameter, 
though  where  crowded  the  spots  usually  coalesce,  forming  diseased 
areas  of  considerable  size. 

"When  only  the  upper  surface  of  leaves  was  subjected  to  the  inocu- 
lating spray  very  few  infections  resulted,  while  inoculations  on  the 
lower  surface  gave  numerous  infections  (PI.  III).  So  far  as  observed 
in  sections  the  infection  takes  place  only  through  stomata,  and 
mostly  from  the  lower  surface.  The  older  and  the  very  young  leaves 
appear  to  be  partially  or  even  completely  immune,  while  those  of 
intermediate  age  (on  the  same  plant)  may  be  seriously  affected. 

The  diseased  leaves  become  yellow  and  fall  off  in  from  three  to  five 
weeks.  The  younger  leaves  and  new  growth  are  healthy.  Under 
our  rather  dry  hothouse  conditions  in  no  case  was  there  evidence  of 
infection  on  any  but  the  inoculated  leaves. 

From  spots  developing  on  leaves  inoculated  with  the  original 
culture  the  organism  was  repeatedly  isolated  by  means  of  petri- 
dish  poured  plates,  and  subcultures  from  colonics  were  again  used 
for  inoculations,  always  with  the  production  of  the  characteristic 
infection. 

The  checks  in  all  cases  remained  healthy;  also  numerous  other 
cauliflower  plants  in  the  same  greenhouse. 

Cabbages  inoculated  with  this  organism  became  infected  in  the 
same  manner  as  the  cauliflower  with  one  exception,  viz,  the  spots 
were  darker.  From  spots  appearing  on  the  inoculated  cabbages  the 
organism  was  isolated  and  tested  on  cauliflower,  with  the  result  of 
the  production  of  the  disease. 

During  May  and  June  of  1909  all  inoculations  resulted  in  successful 
and  typical  infections. 

On  July  10,  1909,  seven  plants  were  inoculated  in  the  usual  manner 
and,  contrary  to  expectations,  no  infections  resulted.  The  conditions 
were  the  same  as  in  previous  inoculations,  except  that  the  tempera- 
ture was  higher  at  this  time,  26°  to  34°  C.  (78°  to  93°  F.).  Subse- 
quent experiments  show  that  the  bacterium  causing  the  disease 
refuses  to  grow  in  artificial  media  at  29°  C.  (84°  F.)  or  above.  It  is 
probable,  therefore,  that  in  this  case  the  high  temperature  prevented 
infection. 

After  July  no  more  plants  were  available  for  inoculation  until  Janu- 
ary 24, 1910,  when  10  plants  were  inoculated.  No  infections  resulted. 
On  February  2,  1910,  10  more  plants  were  inoculated  and  no  infec- 
tions resulted.  All  conditions  seemed  favorable,  and  loss  of  virulence 
in  (he  culture  was  suspected  as  the  cause  of  failure  to  infect.  The 
cultures  used  for  inoculation  were  descended  through  numerous 
transfers  made  during  the  winter.  An  agar-stock  culture  which  had 
not  been  transferred  since  September,  1  ()()(),  was  then  tried.  Fresh 
agar-slant  cultures  were  made  from  the  September  stock  and  used 

225 


DESCRIPTION    OF   TIIK    LEAF-8POT    ORGANISM.  9 

when  24  hours  old  (February  12,  1910)  for  inoculating  cauliflower 
plants.  'I'll is  time  infections  resulted.  The  infections  weret  ery  slight, 
however,  and  the  spots  too  few  in  number  to  cause  any  noticeable 
injury  t"  the  plants,  bu1  the  bacteria  plated  from  these  spots  pro- 
duced typical  colonies  on  agar  and  characteristic  growth  in  artificial 
media.  Cauliflower  plants  inoculated  with  this  new  strain  became 
very  generally  infected,  which  Tact  seem-,  t  .  indicate  that  the  organ- 
ism had  increased  in  virulence  by  passage  through  the  host. 

On  December  6,  L910,  cabbage,  cauliflower,  turnip,  rutabaga,  rad- 
ish, and  mustard  were  inoculated  in  cages  by  spraying  with  sterile 
water  to  which  had  been  added  agar-streak  cultures  :»  days  old.  All 
o!'  these  plants  were  young.  There  were  two  to  lour  of  each  sort. 
The  materia]  for  inoculation  was  obtained  from  a  cauliflower  inocu- 
lated November  _'-'.  1910.  Infections  were  obtained  on  cabbage 
and  cauliflower  (six  days),  but  not  on  the  other  plants.  Sections  of 
the  spots  made  on  the  tenth  day  showed  them  to  be  full  of  bacteria. 

On  March  1,  1911,  three  cauliflower  plants  were  again  inoculated 
h\  spraying  with  sterile  water  to  which  had  been  added  2-day-old 
agar-slanl  cultures.  These  plants  were  about  8  inches  high  and  \m. 
healthy.  They  were  kept  in  an  infection  cage  for  two  days.  At  the 
end  of  ;(i  daj  s  t  here  were  very  small  dark  speck-  on  each  of  I  he  plants. 
These  speck-  were  in  the  center  of  a  small  semil  ransparent  eleval  ion. 
A  microscopic  examination  showed  bacteria  present  m  these -pots. 
'The  flower  stalks  of  plant  101  also  showed  elongated  water-soaked 
spot  s,  darker  in  the  center.  These  also  contained  bacteria  and  plates 
made  from  them  yielded  the  organism  in  pure  culture.  The  flower- 
ing part  of  plant  104  bore  no  spots,  although  it  had  been  drenched 
with  the  spray.      The  check  plant   remained  healthy. 

Several  attempts  to  inoculate  the  head-  of  the  caulillower  gave  no 

satisfactory  results.  Growing  heads  were  copiously  inoculated  and 
kept  under  moist  conditions,  but  no  infections  occurred.  Infection 
spots  similar  to  those  on  leafstalks  and  midribs  occurred  on  some 
of  tin"  larger  stalks  of  the  flower  head,  while  the  flowering  parts 
remained  free  from  infection.  Mature  heads  from  the  market  were 
abo  inoculated,  hut  as  decay  of  the  tender  surface  was  general  in 
the  checks  as  well  as  in  the  inoculated  beads,  the  results  are  not 
conclusn  e. 

DESCRIPTION   OF  THE   CAULIFLOWER    LEAF-SPOT   ORGANISM. 

MORPHOLOGY. 

II rganism  is  a  short  rod.  forming  long  in  some  media. 

Ends  rounded.  Size  from  leaf  I  ..">  to  2.4  "  l>\  0.8  to  0.9  ft.  Size  in 
24-hour  beef-agar  culture,  temperature  20  to  25  C,  1.5  to  •'!  "  by 
0.9 /£.     No  spores  are  produced.     The  organism  is  activelj  motile  by 

225 


10  A    SPOT    DISEASE    OF    CAULIFLOWEK. 

means  of  one  to  five  polar  flagella,  which  are  two  to  three  times  the 
length  of  the  rod.  (Stained  by  Van  Ermengem's  method;  also  by 
Hugh  Williams's  method.)  Motility  occurs  in  most  artificial  media. 
In  beef-bouillon  cultures  grown  and  kept  at  0.5°  to  1.5°  C.  for  four 
months  the  organism  is  still  motile.  Involution  forms  were  found  in 
alkaline  beef  bouillon  (-17  on  Fuller's  scale).  Pseudozoogloeae 
occur  in  Uschinsky's  solution  and  in  acid  beef  bouillon. 

REACTION   TO    STAINS. 

The  organism  does  not  stain  by  Gram.  Modified  Gram,  using 
amyl  alcohol,  gives  a  deep  blue  stain.  It  stains  readily  and  strongly 
with  carbol  fuchsin,  with  an  alcoholic  solution  of  gentian  violet,  and 
with  a  stain  obtained  from  Dr.  Kinyoun  which  contains  methylene 
blue,  silver  nitrate,  azure  I,  and  azure  II.     It  is  not  acid  fast. 

CULTURAL    CHARACTERS. 

Agar  plates  (  +  15  peptonized  beef  bouillon  with  1  per  cent  agar). — 
Tbe  colonies  are  visible  on  the  second  day  as  tiny  white  specks 
(temperature  23°  C). 

In  three  to  four  days  the  colonies  are  1  to  3  millimeters  in  diameter, 
white  (opalescent  in  transmitted  light),  round,  smooth,  flat,  shining, 
and  translucent,  with  edges  entire.  Structure,  under  hand  lens, 
coarsely  granular  with  internal  reticulations.  Buried  colonies  small, 
lens  shaped.  With  age  the  colonies  become  dull  to  dirty  white, 
slightly  irregular  in  shape,  the  edges  undulate,  slightly  crinkled,  and 
with  indistinct  radiating  marginal  lines.  The  internal  reticulations 
disappear  and  the  coarsely  granular  appearance  changes  to  finely 
granular.  In  thinly  sown  plates  7-day-old  colonies  are  6  to  8  milli- 
meters in  diameter;  15-day-old  colonies  are  12  to  15  millimeters  in 
diameter. 

Agar  stabs. — The  surface  of  the  agar  is  covered  in  two  days  (22°  to 
24°  C.)  by  a  thin  white  growth.  For  several  days  the  stab  shows  a 
moderate  growth  in  the  upper  8  to  10  millimeters,  but  this  does  not 
continue.  Finally,  the  stab  is  almost,  if  not  quite,  invisible.  Crystals 
appear  in  the  stab  and  on  the  surface. 

Agar  slants. — In  smear  cultures  the  surface  is  covered  in  two  days 
(temperature  19°  to  21°  C.)  with  a  thin  white  growth,  glistening, 
coarsely  and  irregularly  pitted.     White  sediment  in  the  V. 

In  streak  cultures  in  two  days  (temperature  19°  to  21°  C.)  the 
streak  is  3  to  5  millimeters  wide,  white,  margins  slightly  undulate. 
The  internal  reticulations  seen  in  colonies  on  plates  are  present  in 
the  streak  cultures.  At  right  angles  to  the  streak  are  fine  lines 
extending  from  center  to  margin. 

Agar  cultures  become  slightly  greenish. 

225 


DESCRIPTION    OF   TJIK    l.K  AK-si'oT    <>U<;.\NI8M.  11 

Beef  bouillon. — Peptonized  beef  bouillon  +  l">  held  at  24  to  25  C, 
it'  inoculated  from  young,  vigorous  bouillon  cultures,  clouds  thinly 
in  6  hours  and  is  moderately  to  heavily  clouded  in  24  hours.  The 
growth  is  besl  at  the  surface,  where  a  white  layer  is  formed.  This  is 
not  a  true  pellicle,  as  if  disintegrates  when  the  cultures  are  handled.1 
No  zoogloese  arc  present.  There  is  no  rim.  Tn  two  days  there  are 
heavy  clouds  and  a  moderate  amount  of  white  flocculenl  precipi- 
tate. After  several  weeks  the  precipitate  is  white  and  slimy,  mod- 
erate in  quantity,  and  with  small  crystals  in  it.  The  medium 
becomes  slightly  greenish.  After  several  months  the  precipitate  is 
viscid. 

Acid  bouillon.  -In  neutral  heel'  bouillon  plus  vegetable  acids, 
growth  occurs  until  an  acidity  of  -;:;i  for  oxalic  acid  and  +86  for 
malic  and  citric  acids  (Fuller's  scale)  is  reached.  There  is  no  rim  or 
pellicle.  Occasionally  pseudozoogloeffl  are  formed  in  the  more  acid 
media. 

Microscopic  examination  shows  most  of  the  organisms  greatlj 
reduced  m  length,  some  so  short  as  to  be  spheroidal.  That  these 
were  not  contaminations  was  proved  by  plating  out  and  by  tests  on 
other  culture  media. 

ATkalim  bouillon. — Tn  alkaline  beef  bouillon  (XaOTT  used  >  the 
organism  grew  well  in  —  17.  -  10.  —22,  less  in  —23,  and  not  at  all  in 

25,  -26,  and  28  (Fuller's  scale),  [nvolution  forms  and  filaments 
were  present  in   —17  beef  bouillon  when  two  weeks  old. 

Bouillon  with  sodium  chlorid.  -  In  beef  bouillon  plus  2  percent  XaCl 
the  growth  is  as  good  as  in  plain  beef  bouillon.  With  (he  addition  of 
more  \a(  1  the  growth  gradually  lessens  until  it  is  scarcely  noticeable 
in  a  5  per  cent  solution.  When  grown  in  a  1  per  cent  solution,  the 
'-an  is  not  motile.  In  a  2  per  cent  solution  the  organism  is 
motile,  but  less  so  than  in  beef  bouillon  without  XaCl. 

Beef  bouillon  "<•,/■  chloroform. — For  the.  first  24  hours  the  growth  is 
somewhat  retarded.  By  the  end  of  48  hours  no  difference  could  be 
seen  between  culture-  over  chloroform  (5  c.  c.  of  chloroform  with  10 
c.  c.  of  beef  bouillon  i h >t  shaken)  and  those  in  plain  beef  bouillon. 

Loeffler's  />/"<»/  serum.— Growth  of  stroke  is  moderate,  smooth. 
shining;  color  creamy;  margins  finely  crinkled.  No  liquefaction. 
After  three  months  the  whole  medium  was  slightly  browned. 

Cohn' 8 solution.  -Moderate  clouding  and  white  precipitate:  no  rim, 
pellicle,  or  zoogloese;  no  fluorescence.  After  some  weeks  feather- 
like  crystals  of  considerable  size  (5  to  10  by  2  to  6  mm.)  are  formed. 

/■'<  rm'i's  solution. — Moderate  clouding  at  first.  Precipitate  mode- 
rate to  abundant,  white,  flocculenl.  Pellicle  white,  tender,  sinking 
in  strings  and  masses.     Finally  the  medium  is  densely  clouded  and 

1  Old  cultures  kept  [or  several  mouths  at  0.5°  to  1.5°  C.  had  a  delicate  pellicle. 


12  A    SPOT    DISEASE    OF    CAULIFLOWER. 

pale  green-fluorescent  (between  water  green,  and  greenish  glaucous, 
Kepertoire  de  Couleurs,  Paris,  1905);  more  precipitate  than  in  beef 

bouillon. 

Nutrient  gelatin,  (+10  on  Fuller's  scale). — The  stab  cultures  lique- 
fied  in  8  to  10  days  (temperature  17°  to  18°  C).  Growth  from  sur- 
face crateriform.  Slight,  white,  granular  precipitate.  Slight  green 
fluorescence. 

The  plate  cultures  showed  no  signs  of  growth  in  24  hours  at  17°  to 
18°  C.  In  three  days  well-isolated  colonies  vary  from  mere  points  to 
round  growths  2  millimeters  in  diameter.  The  gelatin  is  liquefied  in 
cuplike  hollows.  Margin  of  smaller  colonies  entire,  of  larger  colonics 
fimbriate.  Thickly  sown  plates  entirely  liquefied  in  two  days  at  15° 
to  16°  C. 

Litmus  milk. — The  medium  becomes  dark  blue  at  the  surface  in 
12  to  24  hours.  The  darkening  proceeds  downward  in  definite  layers 
until  in  8  to  10  days  the  whole  medium  is  dark  blue  with  a  slight  white 
precipitate.  During  six  months'  observation  the  medium  remained 
dark  blue  (reflected  light)  and  liquid.  Finally  by  evaporation  the 
medium  becomes  thickened,  but  there  is  at  no  time  any  separation 
into  curd  and  whey. 

A  few  cultures  showed  a  trace  of  reduction  of  litmus  at  the  bottom. 

Milk. — As  in  the  litmus-milk  cultures,  growth  and  color-change  in 
the  milk  begin  at  the  surface,  proceeding  downward  in  definite  layers. 
In  15  to  20  days  the  whole  tube  (10  c.  c.  of  milk)  is  yellow  (near 
fiidgway's  Naples  yellow,  but  somewhat  duller  and  with  a  greenish 
tinge)  and  translucent.  No  separation  into  curd  and  whey.  Fat 
not  changed.  In  four  months  the  medium  is  quite  dark  (reddish- 
brown)  and  somewhat  thick  (evaporated  to  about  5  c.  c.).  Small 
tyrosin  crystals  are  formed.  These  are  distinctly  visible  only  with 
a  lens. 

Uschinsky's  solution. — Growth  moderate  to  copious;  pellicle  white, 
tender,  breaking  and  sinking  easily.  Pseudozoogloese  are  present. 
There  is  a  greenish  fluorescence.  The  old  cultures  are  much  like 
those  in  Fermi's  solution. 

OTHER    CHARACTERISTICS. 

Ft  r mentation  tubes. — The  organism  is  aerobic  and  does  not  form  gas. 
It  was  tested  in  fermentation  tubes  in  the  presence  of  dextrose, 
saccharose,  lactose,  maltose,  glycerin,  and  mannit,  each  of  these 
carbon  compounds  being  added  to  a  basal  solution  consisting  of  1 
per  cent  of  Wltte's  peptone  dissolved  in  water.  It  did  not  grow  in 
the  closed  end  of  the  fermentation  tubes  in  the  presence  of  any  of 
these  substances. 

Ammonia  'production.     Moderate. 


DESCRIPTION    OF    THE    LEAP-SPOT    ORGANISM.  13 

Nitrates. — Nitrates  are  doI  reduced. 

Indol. —  Iiulol  production  is  feeble. 

Hydrogen  sulphid. — Hydrogep  sulphid  is  not  formed  in  culture-  on 
beef-peptone  agar,  potato  cylinders,  turnip  cylinders,  or  in  beef 
bouillon  or  milk. 

TKMl'Ki:  \  i  I  RE    RE]   vi  CONS. 

'Flu final  death  point. — The  thermal  death  point  is  46°  C.  The 
following  tests  were  made:  Newrj  inoculated  beef-bouillon  (+15) 
cultures  in  tubes  were  suspended  in  a  hot-water  hath  where  they  were 
kepi  for  10  minutes  ai  a  constant  temperature,  then  removed  to  room 
temperature  (20°  to  24°  C).  First,  temperatures  ranging  from  40° 
to  50°  ('.  were  tried,  and,  the  thermal  death  point  seeming  to  lie 
about  halfway  he!  ween,  trials  were  again  made  of  45°,  46°,  and  47° 
C.  More  than  half  of  the  cultures  exposed  to  45°  C.  for  10  minutes 
clouded  in  3  to  5  days.  Of  cultures  exposed  to  46°  C.  1  out 
of  12  clouded  after  1  1  days.  The  others  never  clouded.  Of  20  cul- 
tures exposed  to  -J7°  ('.,  none  clouded. 

Optimum  temperature. — The  optimum  temperature  for  growth  is 
24    to25°C. 

Maximum  temperature. — The  maximum  temperature  for  growth  is 
very  low,  viz,  29°  C. 

Minimum  temperature  'The  minimum  temperature  for  growth  is 
below  0°  C. 

The  organism  was  dead  after  exposure  for  3^  days  at  33  to  36° C. 
in  heel'  houillon. 

EFFECT   OF    DESICCATION. 

When  young,  well-clouded  beef-bouillon  cultures  were  dried  on 
cover  glasses  and  kept  in  a  dark  place  at  temperatures  of  22°  to  25° 
('.,  75  per  cent  were  killed  in  24  hours  and  90  per  cent  in  48  hours. 
All  were  dead  in  five  days. 

EFFECT  OF   SUNLIGHT. 

Four  minutes'  exposure  to  sunlight  killed  all  organisms  in  thinly 
sown  agar  poured  plate-  exposed  bottom  up  on  ice,  one-half  of  each 
plate  being  covered  as  a  check. 

EFFE<   I    OF    1  REEZING. 

Freezing  by  mean-  of  sail  and  pounded  ice  for  two  and  five  hours 
in    +-15  beef  bouillon   had   no  eil'ect    in   reducing  the  number  or  the 

vitality  of  the  organisms,  as  shown  by  poured-plate  cultures  made 

before  and  after  freezing. 

Beef  bouillon  (10  C  C.)  inoculated  and  at  once  frozen  and  kept  at 
temperatures  of   —4°  to   —  1NJ  (.'.   (average   —12°  C.)  for  nine  days 


14  A    SPOT    DISEASE    OF    CAULIFLOWER. 

showed  no  growth  during  this  period,  but  clouded  moderately  three 
days  after  removal  to  temperature  of  18°  to  20°  C,  and  plates  from 
this  tube  gave  pure  cultures  of  the  cauliflower  organism.  Plates 
poured  before  and  after  10  days'  freezing  showed  considerable  reduc- 
tion in  the  numbers  of  organisms,  but  the  growth  of  the  living  ones 
was  not  retarded.  Some  tubes  of  beef  bouillon,  inoculated  with  a 
3-millimeter  loop  from  a  48-hour  bouillon  culture  and  kept  frozen  for 
seven  days,  cloudea  within  48  hours  after  removal  to  a  temperature 
of  18°  to  20°  C.  Another  frozen  for  22  days  did  not  cloud  after 
removal  to  temperatures  of  18°  to  20°  C.  The  check  clouded.  The 
organism  grows  readily  at  low  temperatures,  e.  g.,  beef-bouillon 
cultures  clouded  in  seven  days  when  kept  at  temperatures  of  0°  to  1  °  C. 

VITALITY    ON    CULTURE    MEDIA. 

This  organism  remains  alive  for  six  to  eight  months  at  tempera- 
tures varying  from  18°  to  24°  C.  on  beef  agar,  Loeffler's  blood  serum, 
and  potato  cylinders,  and  in  peptonized  beef  bouillon  (+15),  beef 
gelatin  (  +  10),  milk,  Uschinsky's,  Fermi's,  and  Cohn's  solutions. 
Evaporation  was  not  prevented  in  these  cultures  and  the  media 
became  concentrated,  often  dry,  and  yet  the  organism  was  frequently 
alive.  Beef-agar  and  beef-gelatin  cultures  at  temperatures  of  12°  to 
15°  C.  and  subject  to  less  evaporation  (in  refrigerator)  were  dead  after 
eight  months.  In  media  less  favorable  for  the  growth  of  this  organism, 
as  beef  bouillon  plus  salt,  alkali,  or  acid,  the  bacteria  live  but  two  to 
three  months. 

GROUP   NUMBER. 

The  group  number,  according  to  the  descriptive  chart  of  the  Society 
of  American  Bacteriologists,  is  211.3332023. 

NAME  OF  ORGANISM. 

This  organism  appears  to  be  an  undescribed  form,  and  because  of 
the  characteristic  spotting  of  the  affected  leaves  the  name  Bacterium 
maculicolum  (n.  sp.)  is  suggested. 

LATIN   DIAGNOSIS. 
BACTERIUM    MACULICOLUM   (n.  SP.). 

Baculis  in  hospite  brevibus,  cylindricis,  apicibus  rotundatis,  soli- 
tariis,  saepe  binis  (in  agar-agar  quandoque  10-30  baculis  in  filamenta 
conjunctis);  baculis  1.5-3.0  /*  x  0.8-0.9  [x;  1-5  flagellis  polaribus  mo- 
bilibus;  aerobiis,  asporis. 

Habitat  in  foliis  vivis  Brassicae  oleraceae  in  maculis  1-3  mm.  latis, 
purpureo-griseo  colore.  Coloniae  gelatinam  liquefacientes.  Colo- 
niae  in  agar-agar  rotundae,  albae,  nitentes.  Si  baculi  in  petri-vasibus 
rare  seruntur,  in  septimo  die  colonae  6-8  mm.  diam.  sunt.     Baculi 

225 


SUMMARY.  15 

methodo  Gram  non  colorantur.  Nitrum  uon  redigitur.  Lac  sterile 
alcalinum  fit;  initio  translucidum,  flavum  pallidum  demum  opacum, 
brunnen  e<  gelatum;  casein  non  segregatur.  Enter  temperaturam 
29°  ( '.  el  temporal  uram  -  5°  ( '.  cult  urae  crescunt.  Si  cultural'  iu>\  ae 
in  in  l'i  Kin  in1  carnis  |  horam  in  temporal  ura  16°  C.  tenentur,  moriuntur. 
Enter  temperaturas  —5°  C.  et  — 15°  C.  per  decern  dies  non  moriuntur. 
Si  bacuL'  siccantur  vel  soli  exponuntur,  celeriter  moriuntur.  In  foliis 
vivis  Brassicae  oleraceae  aspergendo  inoculatis,  maculae  in  3  I  diebus 
producuntur.     Contagium  in  stomatibus  fit. 

SUMMARY. 

The  leaf— pot  disease  of  cauliflower  described  in  t  be  preceding  page9 

is  due  to  a  bacterial  organism,  which  was  secured  in  pure  culture  from 
the  leal'  spots  and  inoculated  into  healthy  cauliflower  plants,  with 
production  of  the  disease.  Healthy  cabbages  inoculated  with  the 
organism  also  showed  similar  infection. 

Inoculations  during  July,  1909,  were  unsuccessful  because  of  the 
higher  temporal  are. 

The  heads  of  cauliflower  gave  DO  satisfactory 'results  when  inocu- 
lated wit  li  t  he  organism. 

The  name  BacUnum  dkicuHcoIihh  has  heen  suggested  for  this  organ- 
ism. It  is  a  schizomycete,  pathogenic  to  crucifers,  causing  numerous 
small  --pots  on  cauliflower  and  cabbage.  Entrance  by  way  of  the 
stomata.  Organism  white,  but  causing  a  greenish  fluorescence  in 
some  media  (beef  bouillon  +15,  beef  gelatin  +10,  beef  agar  +15, 
Uschinsky  and  Fermi).  Motile  by  means  of  one  to  several  polar 
Bagella.  A  short  rod  (1.5  to  3  n  by  0.9  /«),  single  or  in  chains  in 
some  media  (10  to  30  >>.  long  on  agar;  10  to  24  fi  long  in  heel'  bouillon 
plus  1  per  cent  sodium  chlorid).  Does  not  stain  by  Gram;  stains 
deeply  with  amyl  Gram. 

No  spores.  Involution  forms  (found  in  alkaline  beef  bouillon)  and 
pseudozoogloese.  Aerobic.  Liquefies  gelatin  slowly.  Does  not 
liquefy  Loeffler's  blood  serum.  Not  gas  forming.  Feeble  produc- 
tion of  ammonia,  indol,  and  hydrogen  sulphid.  Nitrates  no! 
reduced.  Tolerates  acids,  citric  and  malic  to  +36  and  oxalic  to  +34 
(fuller'-  scale).  Tolerates  sodium  hydroxid  in  beef  bouillon  to  —25 
(Fuller's  scale). 

Optimum  temperature  24°  to  25°  C.  Thermal  death  point  46°  C. 
Will   not    grow   in   heel'  bouillon    (+15)   or  on   agar   (+15)   at    1".)°  ('. 

Grows  at  0°  C.  and  below.  Grows  well  in  bouillon  over  chloroform. 
Grows  in  Cohn's  solution.     Blues  litmus  milk. 

The  mos<  si  i  iking  fact  -  about  t  be  organism  are  it-  ahilit  v  to  grow  ,it 
temporal ures  below  freezing  and  its  failure  to  grow  at  a  common  sum- 
mer tempera!  ure  I  85°  F.  I. 

The  leaves  of  the  attacked    plant-  fall  off. 

o 


Bui.  22! 


Plate  I. 


Upper  Surfaces  of  Cauliflower  Leaves  from  Virginia,  Showing   Natural 
Infection  with  Bacterium  maculicolum.    Photographed  May  4,  1909. 


UNIVERSITY  OF  FLORIDA 


3  1262  08928  3229 


